Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen.

Abstract

The purple acid phosphatase from beef spleen, which contains two iron atoms per molecule, is EPR silent in its native (oxidized) purple form. Treatment with mild reducing agents results in conversion to a pink, enzymatically active form, which exhibits an unusual EPR signal centered at g "'1.77; double integration ofthe EPR spectrum gives one spin per two iron atoms. A similar EPR spectrum is observed for enzyme reduced anaerobically by one electron, using sodium dithionite. Variable-temperature magnetic susceptibility measurements show that the oxidized and reduced proteins are both antiferromagnetically coupled systems, with S = 0 and /2 ground states, respectively. Replacement of one of the iron atoms by zinc produces an FeZn enzyme with full catalytic activity. The FeZn enzyme exhibits a highly temperature dependent g = 4.3 EPR signal, and magnetic susceptibility data are consistent with an S = 5/2 paramagnet. Treatment of the FeZn enzyme with phosphate, a competitive inhibitor, results in sharpening of the EPR spectrum; double integration at 77 K gives one spin per iron. These results strongly suggest the presence of a spin-coupled bimetallic unit at the active site of the enzyme. 505 nm) forms, the pink form is produced by treatment of the native purple form with mild reducing agents (1,14). Resonance Raman spectra of the purple form of the porcine enzyme (15) show evidence for coordination of tyrosine phenolate groups [in agreement with similar studies on the sweet potato enzyme (8 16)]. Low-temperature EPR spectra of the porcine enzyme e~hibita novel g' = 1.74 signal, which together with magnetic susceptibility results was interpreted in terms of mononuclear ferric iron in an unusual S = 1/2 spin state in both the purple and pink forms (17).In an attempt to resolve the questions of the nature of the chromophore and its role in catalysis, we have examined the purple acid phosphatase from beef spleen in some detail. We describe herein EPR spectra and magnetic susceptibility data on the native (purple) form, the reduced (pink) form, and a form in which halfthe iron has been replaced by zinc, with complete retention of enzymatic activity. Together with our earlier studies, the present results strongly suggest that the enzyme contains a binuclear spin-coupled iron unit at the active site.Purple metalloproteins with phosphatase activity have now been isolated from mammalian, plant, and microbial sources (1). All but that from Micrococcus sodonensis (2) are similar in being basic glycoproteins with optimal activity at low pH, and all exhibit an intense absorption band at 510-550 nm (E 4,000 M1 cm-') with a shoulder at 310-320 nm. Despite these obvious similarities, there exists substantial disagreement with regard to the metal content. Thus, the protein from beef spleen (3, 4) is reported to contain two iron atoms (5) and that from porcine uterine fluids either one (6) or two (5) iron atoms. The protein from kidney beans apparently contains iron as well (7), although the stoichiometry has not been dete...

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